Glioblastoma multiforme (GBM) remains the most deadly form of brain cancer. There is a dire need to better define the molecular pathways that regulate the malignant features of GBM, in particular, its invasiveness and the persistence of glioma stem cells (GSC), also known as tumor propagating cells, which are thought to be the main source of tumor recurrence due to their tumorigenic potential and therapy resistance. Plexin-B2 is an axon guidance receptor originally identified as a gene highly upregulated in glioma. Our recent patient database analyses found that upregulation of Plexin-B2 correlates with glioma grade and poor survival. This prompted us to investigate the mechanistic basis of this important link. Our studies in human ATCC glioma cell lines demonstrated that Plexin-B2 activation alters actin cytoskeleton and promotes cell migration through activation of Rho GTPases. In intracranial transplants, Plexin-B2 shRNA knockdown hindered invasion and disrupted glioma vascularization. Our new preliminary data from patient-derived GSC lines suggest that besides a promigratory role, Plexin-B2 may also regulate glioma stem cell phenotypes, including stem cell marker expression, self-renewal capacity, proliferation and differentiation potential. Building on these exciting results, we propose to test the central hypothesis that Plexin-B2 plays a multifaceted role in promoting glioma invasion and stem cell characteristics. To facilitate our studies, we have adopted CRISPR gene editing technology to generate bi-allelic mutations of Plexin-B2 in patient-derived GSC lines. In Aim 1, we focus on glioma invasion and ask if Plexin-B2 signaling influences cytoskeletal dynamics and cellular motility of GBM cells by performing culture assays, brain slice cultures, and intracranial transplants. By comparing cell migration of GSCs and their differentiated progeny, this set of studies also addresses whether migratory potential is contingent on differentiation hierarchy. In Aim 2, we focus on glioma stem cell characteristics and ask if Plexin-B2 contributes to stem cell marker expression, self-renewal capacity, proliferation and differentiation potential, and treatment resistance. We will investigate in survival cohorts with GSC intracranial transplants if Plexin-B2 deletion confers survival benefit and if it reduces radiation resistance. In Aim 3, we focus on functional targets and downstream effectors of Plexin-B2 signaling in mediating migration and stem cell properties. We ask which intracellular domain of Plexin-B2 is the main signaling arm, if cellular migration is mediated through modulation of Rho GTPases, whether STAT3 functions as a core signaling hub, and if stem cell factors SOX2 or OLIG2 function downstream of STAT3 for stemness maintenance. Lastly, we will study the adhesion molecules L1CAM and cadherins/protocadherins, the most significantly regulated group of genes that were identified in our recent RNA-Seq analysis of Plexin-B2 knockout GSC. Understanding how Plexin-B2 regulates the malignant features of GBM will open up possibilities for new targets to inhibit glioma growth and invasion.